Nematode control

ABSTRACT

Disclosed herein is a method for controlling nematode populations, particularly soil nematode populations. Certain embodiments comprise applying 4-hydroxybenzylalcohol, Sinapis alba plant extract, Sinapis alba seed meal, or a combination thereof, to soil, optionally in the presence of a trap crop. A hatching factor and/or a nematicide may also be applied to the soil, either substantially simultaneously with the 4-hydroxybenzyl alcohol, Sinapis alba plant extract, Sinapis alba seed meal, or combination thereof, or sequentially in any order. Certain embodiments concern applying 4-hydroxybenzyl alcohol to soil that contains potato cyst nematode eggs. The 4-hydroxybenzyl alcohol may be obtained by an aqueous extraction of Sinapis alba plant material.

CROSS REFERENCE TO RELATED APPLICATION

This is a continuation of U.S. patent application Ser. No. 16/779,846,filed on Feb. 3, 2020, which is a continuation of InternationalApplication No. PCT/US2018/045555, filed Aug. 7, 2018, which waspublished in English under PCT Article 21(2), which in turn claims thebenefit of the earlier filing date of U.S. provisional patentapplication No. 62/544,113, filed Aug. 11, 2017, all of which areincorporated herein by reference in their entireties.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant No.15-8516-1590-CA awarded by United States Department of AgricultureAnimal and Plant Health Inspection Service. The government has certainrights in the invention.

FIELD

Disclosed embodiments concern a method for using of 4-hydroxybenzylalcohol, Sinapis alba plant extract, Sinapis alba seed meal, or acombination thereof, for nematode control.

BACKGROUND

Nematodes are microscopic worms that occur worldwide in mostenvironments. While many species are beneficial to agriculture, somespecies are parasitic to plants, including certain cyst and root-knotnematodes. Cyst nematodes form egg-bearing cysts, typically on plantroots. When the eggs hatch, the nematode invades the root, damaging theplant and reducing the yield of many agricultural crops. Root-knotnematode larvae infect plant roots causing the development of root-knotgalls that can drain the plant's nutrients. If untreated, cysts and eggsmay remain viable in the soil for years, waiting for a suitable hostcrop to be planted, and/or suitable environmental conditions, such astemperature and/or moisture, to occur.

Several commercially important crops that can be impacted by cystnematode infections, including potatoes, soybeans, cereals, such aswheat, and sugar beet.

Nematicides are available to control nematode infections. Those knownnematicides, typically are more effective on live worms, and lesseffective on cysts and eggs. However, most nematicides are syntheticchemicals, such as halogenated fumigants, carbamates, ororganophosphates, that have to be applied to the soil to effectivelycontrol the nematode population. Many of these synthetic nematicides arehighly toxic and many of them are potential environmental pollutants.The use of such synthetic chemicals is incompatible with organicfarming, and the few alternatives available are relatively ineffective.

SUMMARY

Disclosed herein is a method for nematode control, comprising applying4-hydroxybenzyl alcohol, Sinapis alba plant extract, Sinapis alba seedmeal, or a combination thereof, to soil having, or at risk of having,nematode eggs. The 4-hydroxybenzyl alcohol, Sinapis alba plant extract,Sinapis alba seed meal, or combination thereof, is applied in an amountand manner sufficient to effect nematode egg hatch. The 4-hydroxybenzylalcohol, Sinapis alba plant extract, Sinapis alba seed meal, orcombination thereof, may be applied in the presence of a trap crop. Thetrap crop may be any crop suitable for nematode control, such as asolanaceous crop, a cruciferous crop, a grain crop, a tuber-forming,non-Solanum species, or a combination thereof. In some embodiments, thetrap crop is a potato crop, tomato crop, tobacco crop, mustard crop,radish crop, or a combination thereof. The trap crop may be Solanumsysimbriifolium, Solanum aethiopicum, Solanum quitoense, Solanumlycopersicoides, Solanum cercifolium, Brassica juncea, Sinapis alba,Raphanus sativus, Brassica napus, Chenopodium quinoa, Lupinusmutabilius, Ullucus tuberosum, or a combination thereof. In anyembodiments, the 4-hydroxybenzyl alcohol, Sinapis alba plant extract,Sinapsis alba seed meal, or combination thereof, may be applied to thesoil a distance from the trap crop suitable to effect nematode control,such as from greater than zero to 2 feet from the trap crop.

Additionally, or alternatively, the method may comprise applying ahatching factor to the soil optionally obtained from one or more trapcrops, such as a trap crop disclosed herein. The hatching factor maycomprise a potato hatching factor, a potato root diffusate, a tomatoroot diffusate, a soybean root diffusate, a sugar beet root diffusate,or a combination thereof. The hatching factor may be appliedsubstantially simultaneously with the 4-hydroxybenzyl alcohol, Sinapisalba plant extract, Sinapis alba seed meal, or a combination thereof.Alternatively, the hatching factor may be applied from greater than zeroto 6 months or more after application of the 4-hydroxybenzyl alcohol,Sinapis alba plant extract, Sinapis alba seed meal, or a combinationthereof.

Additionally, or alternatively, the method may comprise applying anematicide to the soil. The nematicide may be a carbamate,organophosphate, or a fumigant. The nematicide may be appliedsubstantially simultaneously with the 4-hydroxybenzyl alcohol, Sinapisalba plant extract, Sinapis alba seed meal, or a combination thereof.Alternatively, the nematicide may be applied from greater than zero to 6months or more after application of the 4-hydroxybenzyl alcohol, Sinapisalba plant extract, Sinapis alba seed meal, or a combination thereof.

In any embodiments, the nematode eggs may be encysted eggs. In certainembodiments, the nematode egg is a potato nematode egg, and may be theegg of a potato nematode selected from Globodera achilleae, Globoderaartemisiae, Globodera chaubattia, Globodera ellingtonae, Globoderahypolysi, Globodera leptonepia, Globodera millefolii, Globoderamirabilis, Globodera pallida, Globodera pseudorostochiensis, Globoderarostochiensis, Globodera tabacum, Globodera zelandica, or a combinationthereof. The nematode egg may be a soy nematode egg, such as the egg ofHeterodera glycines. Alternatively, the nematode egg may be a sugar beetnematode egg, such as a Heterodera schachtii egg. In other embodiments,the nematode egg is an egg of a nematode selected from a Globoderaspecies, Heterodera species, Meloidogyne species, Pratylenchus species,Xiphenama species, or a combination thereof.

The method may comprise applying the 4-hydroxybenzyl alcohol, Sinapisalba plant extract, Sinapis alba seed meal, or a combination thereof tothe soil in an amount suitable to facilitate nematode control in thesoil in combination with a trap crop, nematicide, hatching factor, orcombination thereof. Sinapis alba seed meal may be administered in anamount of from 500 lbs meal/acre to 4,000 lbs or more meal/acre, such asfrom 1,000 lbs meal/acre to 3,000 lbs meal/acre, from 1,500 lbsmeal/acre to 2,500 lbs meal/acre, or about 2,000 lbs meal/acre. Sinapisalba plant extract may be administered in an amount of from 50 lbs/acreor less to 1,000 lbs or more extract/acre, such as from 100 lbsextract/acre to 1,000 lbs extract/acre, from 200 lbs extract/acre to 800lbs extract/acre, from 350 lbs extract/acre to 650 lbs extract/acre, orabout 500 lbs extract/acre. 4-hydroxybenzyl alcohol may be applied tothe soil in an amount of from greater than zero to 100 lbs or more peracre, such as from 1 lb/acre to 100 lbs/acre, from 5 lbs/acre to 75lbs/acre, from 20 lbs/acre to 50 lbs/acre, or from 25 lbs/acre to 40lbs/acre. In some embodiments, about 30 lbs/acre 4-hydroxybenzyl alcoholis applied to the soil.

In particular embodiments, the method comprises applying 4-hydroxybenzylalcohol to the soil. The 4-hydroxybenzyl alcohol may be applied in thepresence of a trap crop. Additionally, or alternatively, method maycomprise applying a hatching factor to the soil, applying a nematicideto the soil, or applying both a nematicide and a hatching factor to thesoil, substantially simultaneously, or sequentially in any order.

In any embodiments, the method may comprise applying a formulationcomprising a concentration of 4-hydroxybenzyl alcohol of from greaterthan zero to 6,000 μmol/mL, such as from 100 μmol/mL to 1,000 μmol/mL.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of glucosinolate concentrations (μmol glucosinolatesper gram seed meal determined without using a response factor) forvarious plant materials as determined using hot water (ht) and methanol(me) extractions. Athena and Dwarf Essex are B. napus species, Ida is aS. alba species, and Pac Gold is a B. juncea species. Ida samples m4 andm5 represent two different S. alba meal samples.

FIG. 2 provides first-order plots for the disappearance of4-hydroxybenzyl isothiocyanate incubated in buffered aqueous solutionswith pH values ranging from 3.0 to 6.5, where plots for pH 3.0 and 3.5are superimposed on each other in the graph.

FIG. 3 is a graph of glucosinolate concentrations (μmol glucosinolatesper gram seed meal determined without using a response factor) forvarious plant materials to compare total glucosinolates in stored (old)or freshly pressed (new) meals. Athena and Dwarf Essex are B. napusspecies, IdaGold is a S. alba species, and Pac Gold is a B. junceaspecies.

FIG. 4 is a plot of pH of hydrolysate and percentage of sinalbinhydrolyzed (%) versus expected total concentration (mM), illustratingthe hydrolysis of S. alba mustard extract (0.05-0.3 g) in the presenceof mustard meal (0.1 g) in 2.5 mL of water.

FIG. 5 is a plot of pH of hydrolysate and percentage (%) of sinalbinhydrolyzed versus expected total concentration (mM), illustrating thehydrolysis of B. juncea mustard extract (0.05-0.3 g) in the presence ofmustard meal (0.1 g) in 2.5 mL of water.

FIG. 6 is a plot of glucosinolate hydrolyzed (%) versus pH, illustratingthe hydrolysis of S. alba and B. juncea mustard extracts (0.15 g) in thepresence of mustard meal (0.1 g) in 2.5 mL of 200 mM phosphate buffer.

FIG. 7 is a plot of mass balance closure (%) versus ascorbateconcentration (mM), illustrating the effect of ascorbate addition on theproduction of allyl isothiocyanate and ionic thiocyanate from B. junceaand S. alba mustard powder.

FIG. 8 is a plot of sinalbin hydrolysis (%) versus time (hours),illustrating the hydrolysis of S. alba and B. juncea mustard extracts(0.15 g) in the presence of mustard meal (0.1 g) in 2.5 mL of 150 mMphosphate buffer or 150 mM potassium bicarbonate.

FIG. 9 illustrates the production of ionic thiocyanate (μmol/gram seedmeal) versus time (hours), illustrating production of ionic thiocyanatefrom S. alba seed meal incubated in deionized water and aqueoussolutions buffered at pH values ranging from 4.0 to 7.0.

FIG. 10 is a plot of continuous and periodic extraction of4-hydroxybenzyl isothiocyanate (μmol/gram seed meal) versus time(hours), showing continuous and periodic extraction into ethyl acetateof 4-hydroxybenzyl isothiocyanate resulting from hydrolysis of 4-OHbenzyl glucosinolate contained in S. alba seed meal as compared tosimilar extractions of benzyl isothiocyanate from aqueous solution.4-Hydroxybenzyl isothiocyanate incubations contained no seed meal, butare expressed on a weight basis for comparison purposes only.

FIG. 11 is a graph of Globodera pallida hatch (%) versus treatment type,illustrating the percent Globodera pallida hatch after a 2-week exposureto Sinapis alba seed meal in either potato root diffusate (PRD) or insoil extract.

FIG. 12 is a graph of Globodera pallida hatch (%) versus treatment type,illustrating the percent Globodera pallida hatch after a 2-week exposureto Sinapis alba seed meal, its hydrolysis product 4-hydroxybenzylalcohol (HBA), or to a non-treated bare soil control in potato rootdiffusate (PRD) or in soil extract.

FIG. 13 is a graph of Globodera pallida emergence (%) versus treatmenttype, illustrating the percent hatch of Globodera pallida eggs after a2-week exposure to 5 rates of 4-hydroxybenzyl alcohol (HBA) (0 μmol/ml,1 μmol/ml, 2 μmol/ml, 4 μmol/ml, 8 μmol/m1) in either potato rootdiffusate (PRD) or soil extract.

DETAILED DESCRIPTION I. Definitions

The following explanations of terms and abbreviations are provided tobetter describe the present disclosure and to guide those of ordinaryskill in the art in the practice of the present disclosure. As usedherein, “comprising” means “including” and the singular forms “a” or“an” or “the” include plural references unless the context clearlydictates otherwise. The term “or” refers to a single element of statedalternative elements or a combination of two or more elements, unlessthe context clearly indicates otherwise.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting. Other features of thedisclosure are apparent from the following detailed description and theclaims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, percentages, temperatures, times, and soforth, as used in the specification or claims are to be understood asbeing modified by the term “about.” Accordingly, unless otherwiseindicated, implicitly or explicitly, the numerical parameters set forthare approximations that may depend on the desired properties soughtand/or limits of detection under standard test conditions/methods. Whendirectly and explicitly distinguishing embodiments from discussed priorart, the embodiment numbers are not approximates unless the word “about”is recited.

Cyst: A casing around the eggs of a cyst nematode. Typically the cyst isformed by the body of the female, which instead of laying the eggs,keeps them in her body. After death, the body becomes hard or tanned andprotects the eggs from adverse environmental conditions. Eggs in cystscan remain viable for several years. Approximately 20 to 60 percent ofthe eggs hatch annually under suitable temperature and moistureconditions.

Gall: A swelling growth on the external tissues of plants. Root-knotnematodes typically cause galls to appear on the roots of susceptibleplants.

II. Overview

Nematodes are microscopic worms that are found in almost everyenvironment in the world. While many nematode species are not harmful,and some are even beneficial, certain nematode species attach and feedon plants including crops species. Such attacks can cause substantialcrop yield reductions and associated financial losses for farmers.Disclosed herein is a method for nematode control, comprising applying4-hydrozybenzyl alcohol to soil, particularly soil that contains, or isat risk of containing, nematode eggs. The 4-hydroxybenzyl alcohol may beapplied to soil in the presence of a trap crop, and/or the method mayfurther comprise applying a hatching factor and/or a nematicide to thesoil.

III. 4-Hydroxybenzyl Alcohol

4-Hydroxybenzyl alcohol has a structure

4-Hydroxybenzyl alcohol can be made synthetically, or it can be obtainedfrom plant material, such as Sinapis alba. In some embodiments,4-hydroxybenzyl alcohol is obtained from an aqueous extraction ofSinapis alba plant material, such as seed meal. 4-Hydroxybenzyl alcoholis produced by hydrolysis of the primary glucosinolate in Sinapis alba.The hydrolysis typically is enzymatic hydrolysis that produces4-hydroxybenzyl isothiocyanate that subsequently decomposes in aqueousconditions to form 4-hydrozybenzyl alcohol.

Glucosinolates, found in dicotyledonous plants, are a class of organicanions usually isolated as potassium or sodium salts, but occasionallyin other forms. For example, p-hydroxybenzyl glucosinolate is isolatedas a salt complex with sinapine, an organic cation derived from choline.Features common to the class are a β-D-thioglucose moiety, a sulfateattached through a C═N bond (sulfonated oxime), and a side group(designated R) that distinguishes one glucosinolate from another. Ageneral formula for glucosinolates is provided below.

More than 130 different R groups, and thus glucosinolates, have beenidentified or inferred from degradative products.

Glucosinolate types in plant species are highly variable. For example,the main glucosinolate in radish seed (Raphanus sativus) is4-methylsulphinyl-3-butenyl glucosinolate, while mustard seed (Brassicajuncea) is dominated by 2-propenyl glucosinolate. Cabbage seed (Brassicaoleracea) contains mainly 2-propenyl and 2-hydroxy-3-butenylglucosinolate. Rapeseed (Brassica napus) contains 4 majorglucosinolates: 2-hydroxy-3-butenyl, 3-butenyl, 4-pentenyl, and2-hydroxy pentenyl glucosinolates. Similar differences in glucosinolatetypes are observed when comparing vegetative plant parts.

Brassica and Sinapis species, and many other members of the Brassicaceaeplant family, produce glucosinolate compounds, which are secondarymetabolites. Thus, disclosed embodiments of the present application mayalso comprise determining plants that produce glucosinolates in amountseffective for use as a biopesticide, such as a nematicide, and/or thatcan enhance nematode hatch when used in combination with a hatchingfactor. Glucosinolates are compounds that occur in agronomicallyimportant crops and may represent a viable source of allelochemiccontrol for various soil-borne plant pests. Glucosinolates can beextracted from plant material using aqueous extractions, using polarorganic compounds, such as lower alkyl alcohols as the solvent, or byusing aqueous mixtures of polar organic compounds to performextractions, as illustrated by FIG. 1 .

Glucosinolates are normally stored within plant tissues. Toxicity is notattributed to intact glucosinolates. Upon tissue damage, plant enzymestrigger glucosinolate hydrolysis to several compounds includingnitriles, isothiocyanates (ITCs, —N═C═S), organic cyanides,oxazolidinethiones (OZTs), and ionic thiocyanate (SCN⁻), that arereleased upon enzymatic degradation in the presence of water asindicated in Scheme 1. Degradation also occurs thermally or by acidhydrolysis. Toxicity is generally attributed to these bioactiveproducts.

Myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) is not properlyidentified as a single enzyme, but rather as a family or group ofsimilar-acting enzymes. Multiple forms of the enzymes exist, both amongspecies and within a single plant, and all perform a similar function.Although their genetic sequences are similar to other β-glycosidases,myrosinases are fairly specific toward glucosinolates. These enzymescleave the sulfur-glucose bond regardless of either the enzyme orsubstrate source. However, the particular enzyme and glucosinolatesubstrate influence reaction kinetics.

Myrosinase and glucosinolates are separated from each other in intactplant tissues. Glucosinolates are probably contained in vacuoles ofvarious types of cells. In contrast, myrosinase is contained only withinstructures called myrosin grains, which are specialized myrosin cellsthat are distributed among other cells of the plant tissue. Incold-pressed meal, myrosinase and glucosinolates are no longerphysically separated, and myrosinase activity is preserved. Thus, addingwater immediately results in the production of the hydrolysis products,including isothiocyanate, without the need for additional tissuemaceration.

Nitrile character is common to four additional products. Forming anitrile (R—C≡N, also known as an organic cyanide), which does notrequire rearrangement, involves sulfur loss from the molecule. Nitrileformation is favored over ITC at low pH, and even occurs in somecrucifers at a pH where ITC is normally the dominant product. Thepresence of Fe²⁺ or thiol compounds increases the likelihood of nitrileformation and decreases the proportion of SCN⁻ production.

Epithionitrile formation requires the same conditions as for nitriles,plus terminal unsaturation of the R-group and the presence of anepithiospecifier protein. The epithiospecifier protein possesses a rareproperty in that it is an enzyme cofactor that allosterically directs anenzyme to yield a different product. Thiocyanate (R—S—C≡N) is sometimesproduced, particularly in members of the Alyssum, Coronopus, Lepidium,and Thlaspi families. Factors controlling organic thiocyanate formationare not well understood.

SCN⁻ production from glucosinolates is controlled by the presence ofspecific R-groups. Evidence suggests the anion is a resonance hybridwith greater charge on the S; however, charge can be localized on eitherthe sulfur (⁻S—C≡N) or the nitrogen (S═C═N⁻), depending on theenvironment. Indole and 4-hydroxybenzyl glucosinolates yield SCN⁻ thatis thought to arise from a highly unstable ITC intermediate. SCN⁻ isformed from indole glucosinolates over a wide pH range, whereas4-hydroxybenzyl glucosinolates typically yield SCN⁻ only at a more basicpH. As discussed below, 4-hydroxybenzyl isothiocyanate is not stableeven at pH values of 3.0, but instead forms 4-hydroxybenzyl alcohol. Thehalf-life decreases with an increase in pH from 3.6 hours at pH 3.0 toless than 5 minutes at pH 7.0 (FIG. 2 ).

ITCs historically have been considered the ‘normal’ products ofglucosinolate breakdown. They often are volatile with pungent flavors orodors. Some of the hydrolysis products, like ITCs, exhibit biocidalproperties on insects, nematodes, fungi and/or weeds. ITC formationrequires that the initial unstable aglucon intermediate undergo aLoessen rearrangement to the R-NCS configuration. Isothiocyanates arequite reactive, although less so than the related isocyanates (R—N═C═O).A few commercially available soil fumigants depend on the activity ofmethyl ITC either as the parent compound or as produced from precursorssuch as sodium N-methyldithiocarbamate ortetrahydro-3,5-dimethyl-2H-1,3,5-thiadiazine-2-thione. Because of knowntoxicities, ITCs are often considered likely candidates for pesticidalactivity.

For Sinapis alba, the glucosinolate precursor to bioactive compounds is4-hydroxybenzyl glucosinolate. Thus, the amount of this compound foundin plants provides another basis for determining plant material usefulfor practicing embodiments of the disclosed invention. The structuralformula for 4-hydroxybenzyl glucosinolate is provided below.

A person of ordinary skill in the art will appreciate that certainderivatives of 4-hydroxybenzyl glucosinolate also potentially may beuseful for practicing disclosed embodiments of the present invention.For example, naturally occurring or synthetic derivatives may includeplural hydroxyl groups, as opposed to the single hydroxyl group presentat the 4 position in 4-hydroxybenzyl glucosinolate. Such derivativesmight have a chemical formula

where one or more of R¹, R², R³ and R⁴ optionally are hydroxyl groups.It also will be appreciated that the hydroxyl groups present in4-hydroxybenzyl glucosinolate, or derivatives thereof, may be present insome other form, such as a protected form, that produces the desiredhydroxyl groups, such as by hydrolysis or enzymatic cleavage. Moreover,halide derivatives also may be useful. As a result, one or more of R¹,R², R³ and R⁴ optionally may be a halide.

The concentrations of 4-hydroxybenzyl glucosinolate in plant materialcorrespond to the amounts of ionic thiocyanate (SCN⁻) produced by suchmaterials. Guidelines for Glucosinolate Analysis in Green Tissues forBiofumigation, Agroindustria, Vol. 3, No. 3 (2004), which isincorporated herein by reference, provides standardized methodology usedto quantitatively determine amounts of such bioactive compounds. Thispublication discusses modifications of the ISO 9167-1 method, initiallyset up for evaluating rapeseed seeds, with the objective of optimizingand standardizing glucosinolate analysis in fresh tissues (leaves, rootsor stems) of Brassicaceae. Collection, storage and preparation of freshsamples suitable to be analyzed are important steps during which it isnecessary to avoid glucosinolate hydrolysis by the endogenousmyrosinase-catalyzed reaction. Differences in glucosinolateconcentrations in stored, processed and fresh meal are illustrated byFIG. 3 .

4-hydroxybenzyl glucosinolate concentrations may be determined usingHPLC/MS. Additional information concerning determining glucosinolateconcentrations is provided below in the working examples, using aninternal standard, such as 4-methoxy benzyl glucosinolate. In summary,the concentration of the 4-hydroxybenzyl glucosinolate is measured, suchas by determining the area under the appropriate HPLC peak. Theconcentration is multiplied by a response factor of 0.5 relative to2-propenyl glucosinolate to determine the concentration of4-hydroxybenzyl glucosinolate.

Certain embodiments concern plant material having effective amounts of4-hyroxybenzyl glucosinolate. The glucosinolate concentration typicallyis determined after plant material has been cold pressed to remove amajority of the plant oil. Residual oil contents for cold pressed plantstypically range from substantially 0% to about 15%, more typically from7% to 12%. If solvent extraction is used for oil removal, oil contentsmay be less than 1%. Glucosinolate concentrations may vary within plantsof a single species, and concentration fluctuations may occur within aparticular plant. Additional environmental factors such as spacing,moisture regime, and nutrient availability also may affectconcentration. Nevertheless, useful 4-hydroxybenzyl glucosinolateamounts are from about 10 μmol/gram to about 500 μmol/gram, typicallyfrom about 10 μmol/gram to about 400 μmol/gram, more typically fromabout 50 μmol/gram to about 250 μmol/gram, and even more typically fromabout 75 μmol/gram to about 210 μmol/gram.

Portions of plant material, leaves, stems, roots and seeds that have thehighest concentration of glucosinolate commonly are used to practiceembodiments of the disclosed process. Meal is preferably made fromseeds; however it is possible to use any plant material containingglucosinolate to make the meal. For example, with reference to theexemplary Sinapis alba plant material, it has been found that Sinapisalba seeds contain the highest levels of 4-hydroxybenzyl glucosinolate.Sinapis alba is useful for making biodiesel. In one embodiment,biodiesel production crushes the seeds to liberate the oil, leaving theseed meal as a by-product. This by-product had limited use prior todevelopment of the present invention. The seed meal now can be used topractice embodiments of the presently disclosed process.

In some embodiments, glucosinolates are extracted from the plantmaterial or processed plant material, such as seed meal produced by theproduction of biodiesel. Extracting glucosolinolates from the plantmaterial or processed plant material has several advantages. It cansignificantly reduce the effects of batch-to-batch variability resultingfrom variability in plant growing conditions, processing and storage.Also, the cost and logistics of transportation, storage, and applicationof mustard meal are relatively high compared to extracts of the same.And the introduction of large amounts of meal to the soil may result ina large organic carbon load (mustard contains up to 80% of organiccarbon by weight), which can create some adverse effects such as growthof undesirable microorganisms.

Additionally, the extracts are typically stable in storage for at leastthree months at 25° C., and up to a year or more at −4° C. The extractsare typically not light sensitive, are thermally stable up to 120° C.,and do not promote mold growth. In some embodiments, the extracts mayhave at least twice the concentration of active ingredients as mustardmeal, such as up to three times, up to four times, or more than fourtimes the concentration. The extracts can be prepared either as apowder, or as a solution in variety of active agent concentrations asrequired for different applications. The use of such solutions makes theextracts compatible with spray delivery systems.

The plant material or processed plant material is extracted using asolvent system suitable for extracting glucosinolates from the plantmaterial. The solvent system used for the extraction may be a singlesolvent or mixture of solvents. Typically, an aqueous solvent system isused for the extraction, such as a solvent system comprising water andoptionally an alkyl alcohol. The alkyl alcohols may comprise one or moreC₁-C₄ alkyl alcohols, such as methanol, ethanol, propanol, isopropanol,n-butanol, isobutanol, sec-butanol or tert-butanol. In some embodiments,a single alcohol is used, but in other embodiments, two or more alcoholsare used, such as a mixture of methanol and ethanol.

The solvent system may comprise from greater than 0% to 100% water andfrom less than 100% to 0% alcohol, such as 100% water, from 10% to 90%water and from 90% to 10% alcohol, or from 30% to 70% water and from 70%to 30% alcohol. In some embodiments, a ratio of water to alcohol isselected to inhibit or substantially prevent the glucosinolates fromhydrolyzing during the extraction. In such embodiments, the percentageof alcohol in the solvent system is from 60% to less than 100%, such asfrom 65% to 95% or from 70% to 90%. In certain embodiments, seed meal,such as B. juncea seed meal, is extracted with an extraction solventcomprising, consisting essentially of, or consisting of from 60% to lessthan 100% alcohol and from greater than zero to 40% water, such as from60% to 90% alcohol and from 10% to 40% water, from 60% to 80% alcoholand from 20% to 40% water, or from 65% to 75% alcohol and from 25% to35% water, and in particular embodiments, the seed meal is extractedwith an extraction solvent comprising, consisting essentially of, orconsisting of about 70% alcohol and 30% water. In certain embodiments,the alcohol is ethanol.

Alternatively, the ratio of water-to-alcohol may be selected to promotehydrolysis of the glucosinolates to form 4-hydroxybenzyl alcohol duringthe extraction. In such embodiments, the solvent system typicallycomprises an excess of water. The solvent system may be 100% water, orit may comprise from 60% to 100% water and from 40% to 0% alcohol, suchas from 70% to 90% water and from 30% to 10% alcohol. The extractionprocess may continue for a time period suitable to allow hydrolysis ofthe glucosinolates. In some embodiments, the extraction is performed forup to 5 days, such as up to 3 days, or from 2 to 3 days, to allow forextraction and hydrolysis to take place. The extraction may be performedat a temperature suitable to facilitate the extraction, such as fromgreater than zero to 80° C. or more, from 5° C. to 70° C., or from 20°C. to 40° C. During the extraction the pH optionally may be buffered,such as to maintain a pH of from 3.0 to 10.0, typically from 5.0 to 7.0.Examples of suitable buffers include, but are not limited to, phosphate,carbonate, bicarbonate buffers or combinations thereof.

Mustard meal can absorb water and swell up by up to about 400% or more.Accordingly, in most embodiments, the amount of mustard meal added tothe aqueous solution is from greater than zero to 25% by weight,typically up to 20% by weight, to facilitate recovery of the liquid. Insome embodiments, the amount of mustard meal is further limited to allowfor reasonable recovery of the desired products, and may be 10% or lessby weight, such as 7% or less or 4% or less.

The extracts may be filtered to remove any solid material, and thenevaporated by any suitable technique known to a person of ordinary skillin the art, to remove the extraction solvent(s). Suitable techniquesinclude, but are not limited to, rotary evaporation, optionally undervacuum, spray drying, belt drying, drum drying, freeze drying or anycombination thereof. In certain embodiments, spray drying is preferred.Typically, the evaporation and/or drying will produce a solid extract,which may be in the form of a powder, such as a free-flowing powder.

Glucosinolates themselves are not biologically active and can bepreserved in extracts for a substantial period of time, such as fromgreater than zero to 3 years or more. However, in the presence of water,glucosinolates are converted by the endogenous enzyme myrosinase(thioglucoside glucohydrolase, EC 3.2.1.147) into biologically activecompounds. The major glucosinolate in S. alba, sinalbin, is hydrolyzedto an unstable isothiocyanate that non-enzymatically produces SCN⁻, aphytotoxic compound, and 4-hydroxybenzyl alcohol (4-hydroxybenzylalcohol) (Scheme 2). The major glucosinolate in B. juncea, sinigrin, ishydrolyzed to produce a volatile, bioactive 2-propenyl isothiocyanate,that may be used as a nematicide.

Myrosinase is present in mustard meal, such as S. alba and B. junceameal. Thus, mustard meal may be added to an aqueous solution or to apowdered extract of glucosinolates to aid hydrolysis. The aqueoussolution or powdered extract also may comprise a buffer, such as aphosphate buffer, carbonate buffer, bicarbonate buffer, or a combinationthereof, to maintain a pH preferable for the activity of the enzyme. Insome embodiments, the pH is from about 5 to about 8, such as from 6 to7.5.

Mustard extract hydrolysis can be performed prior to applying extract tothe field. For example, a S. alba extract containing sinalbin can behydrolyzed to yield 4-hydroxybenzyl alcohol. This solution can beapplied through existing sprinkler or sprayer systems. Alternatively,mustard extract hydrolysis can be performed in situ by applying mustardextract to the field and hydrolyzing directly at the point of pestcontrol. This approach is particularly useful for pest and nematodecontrol when using the volatile, allyl isothiocyanate hydrolysis productof sinigrin from B. juncea.

IV. Nematodes

Nematodes are microscopic worms representing the most numerousmulticellular animals on Earth. Some nematodes are plant parasites thatattack agronomically important crops, causing decreases in yield andquality. Many plant parasitic nematodes require a specific host forsuccessful reproduction. When a suitable host is not present, plantparasitic nematodes may remain dormant in soil as free-living eggs oreggs contained in a protective cyst, inactive but viable for years. Theeggs may be stimulated to hatch by, for example, suitable environmentalconditions, such as temperature and/or moisture levels; and/or asuitable plant host growing within a suitable distance. Certain eggs,particularly, encysted eggs, are stimulated to hatch by the release ofchemicals from plants. These chemicals are referred to as hatchingfactors. Hatch factors can be released by either a host plant requiredfor reproduction or a closely related non-host species. Hatching factorsalso can be added artificially to stimulate nematode hatch.

Certain soil characteristics may be beneficial for nematode growth. Insome embodiments, the soil has a pH of from 4 or less to 9.5 or more,such as from 4 to 9.5, from 4.5 to 9, or from 4.5 to 8.5. The organiccarbon concentration of the soil may be from greater than zero to 45% ormore, such as from 0.1% to 45%, or from 0.5% to 10%. The soil maycomprise from 0 to 90% clay, from 0 to 90% silt, from 0 to 90% sand, ora combination thereof. In some embodiments, the soil comprises from 10%to 50% clay, from 10% to 50% silt, from 10% to 50% sand, or acombination thereof. Typically, soil temperatures above 4° C. and below40° C. are preferred for hatch, more typically 10° C. to 25° C. Moisturecontent on a weight basis of 5% to 50% is preferred for hatch, moretypically 10% to 20%.

Certain parasitic nematodes infect commercially important agriculturalcrops, including potato, soybean, cereal, such as wheat, and sugar beetcrops. With respect to potatoes, the potato cyst nematode causes potatogrowth retardation, reducing yields by 60% or more in areas of highnematode populations.

The potato cyst nematode belongs to the genus Globodera, which includesabout 12 species: Globodera achilleae; Globodera artemisiae; Globoderachaubattia; Globodera ellingtonae; Globodera hypolysi; Globoderaleptonepia; Globodera Globodera mirabilis; Globodera pallida; Globoderapseudorostochiensis; Globodera rostochiensis; Globodera tabacum; andGlobodera zelandica. The nematodes live on the roots of plants of theSolanaceae family, which includes potatoes, tomatoes, eggplant, bellpeppers, chilli peppers, and tobacco. Nematodes of particular interestinclude Globodera rostochiensis on potato, and Globodera tabacum ontobacco.

The cyst nematode genus Heterodera includes at least 70 species. Amongthese are several species that are parasitic to commercial crops. Forexample, the soybean cyst nematode, Heterodera glycines, is the mostserious soybean pathogen in the world. Since its discovery in the UnitedStates in 1954, it has spread to all states with significant soybeanacreages. Severe yield loss caused by this pathogen is especially commonin sandy soils. Soybean cyst nematode, however, is not restricted to anysoil type and often causes significant soybean yield losses, which maygo unnoticed, even in clay soils.

The sugar beet cyst nematode, Heterodera schachtii, can infect more than200 plant species, including sugar beet, garden beet, table beet andcanola, causing the infected plants to become pale yellow and wilt. Thenematode can also survive on common weeds, such as wild mustard,pigweed, lambsquarters, shepherdspurse and purslane. Sugar beet cystnematodes are found worldwide, and in the United States are present inalmost all sugar beet-producing states. In heavily infected areas,losses due to sugar beet cyst nematode infections can be as much as $750per acre.

Exemplary Heterodera species include, but are not limited to, Heteroderacarotae, Heterodera cruciferae, Heterodera humuh, Heterodera trifolii,Heterodera galeopsidis, Heterodera goettingiana, Heterodera betae,Heterodera sacchari, Heterodera cajani, Heterodera glycines, orHeterodera schachtii. Other exemplary Heterodera species are sometimesgrouped together as the “Heterodera avenae group.” The Heterodera groupmay include Heterodera avenae, Heterodera fihpjevi, Heterodera arenaria,Heterodera aucklandica, Heterodera bifenestra, Heterodera hordecalis,Heterodera iri, Heterodera latipons, Heterodera mani, Heteroderapratensis, Heterodera spinicauda, and/or Heterodera turcomanica. TheHeterodera avenae group, or cereal cyst nematodes, invade and reproduceonly in living roots of cereals and grasses, but not broadleaf plants.Damage from cereal nematode infections can be similar to symptomsassociated with irregularities in soil depth, texture and/or pH, mineraland/or water availability, or other diseases. Accordingly, cerealnematode infections are routinely underestimated, but it is nowestimated that nematode infections in the wheat crops of Idaho, Oregonand Washington states alone may reduce the wheat profitability by atleast $3.4 million annually.

Other nematode species include, but are not limited, to root-knotnematodes, such as Meloidogyne spp. including M. incognita, M. hapla, M.javanica, M. chitwoodi, M. arenaria, M. naasi, M. acronea, M. artiellia,M. brevicauda, M. coffeicola, M. exigua, M. gajuscus, M. enterolobii, M.partityla, M. thamesi or M. fallax; lesion nematodes, such asPratylenchus spp. including Pratylenchus alleni, Pratylenchusbrachyurus, Pratylenchus coffeae, Pratylenchus crenatus, Pratylenchusdulscus, Pratylenchus fallax, Pratylenchus flakkensis, Pratylenchusgoodeyi, Pratylenchus hexincisus, Pratylenchus loosi, Pratylenchusminutus, Pratylenchus mulchandi, Pratylenchus musicola, Pratylenchusneglectus, Pratylenchus penetrans, Pratylenchus pratensis, Pratylenchusrenifonnia, Pratylenchus scribneri, Pratylenchus thornei, Pratylenchusvulnus, or Pratylenchus zeae; and dagger nematodes, such as Xiphenamaspp. including X. americanum, X. diversicaudatum, X. index, X. italiae,X. bakeri, X. brevicolle, X. insigne, X. rivesi, X. vuittenezi, X.brasiliense, X. hygrophylum, X. stenocephalum, X elongatum, X. coxi, X.ingens, X. conurum, X. pachydermum, X. mammallatum, X. thorneanum, X.mehtense, X. bacaniboia, X. surinamense, X. guirani, X. porosum, X.rotundatum, X. spinuterus, X. bergeri, X. douceti, X. nigeriense, X.attarodorum, X. clavicaudatum, X. elongatum, X. ifacolum, X.longidoroides, X. setariae, X. ifacolum, X. ebriense, X. pini, X.turcicum, X. basiria, X. marsupilami, X. malagasi, X. radicicola, X.longicaudatum, X. krugi, X. costaricense, X. filicaudatum, or Xpachtaicum; or any combination thereof.

V. Hatching Factor

Hatching factors, or hatch factors, are chemicals released by plantsthat stimulate nematode eggs, including encysted eggs, to hatch.Hatching factors may be obtained from plant root diffusates orleachates, and a single diffusate may contain more than one hatchingfactor. For example, it has been shown that potato root leachate maycontain at least ten hatching factors. The plants typically are thespecific plants, or hosts, required for reproduction, or a closelyrelated non-host plant species. Both potato and tomato root diffusatecan be used as a hatching factor for potato cyst nematodes.

In some embodiments, a single hatching factor is administered, but inother embodiments, multiple hatching factors, such as 2, 3, 4, 5, 6, 7,8, 9, 10 or more hatching factors, are administered eithersimultaneously or sequentially in any order. And/or a root diffusatefrom one or more plant species may be administered simultaneously orsequentially in any order, such as 1, 2, 3, 4, 5 or more different rootdiffusates, each comprising one or more hatching factors.

A. Trap Crops

Non-host species are often used to control plant parasitic nematodes bystimulating cyst hatching, thereby producing a nematode that dieswithout reproducing. These non-host species are referred to as trapcrops. The potential threat to future host crops planted in that samefield is thus reduced or eliminated. While the use of crop rotationswith trap crops to break the growth cycle is one of the simplest andmost cost-effective ways of controlling nematodes, hatch stimulation bythe trap crop is typically incomplete and many encysted eggs remainviable.

Crucifer crops have a good reputation for rotating with cereals, sugarbeets and potatoes. They have deep taproots, which break up plow pans,improve soil tilth and they are a good supplier of organic matter.Historically, mustards and radishes have been good nematode hosts,particularly for the beet cyst nematode. As a result, using mustards andradishes in a rotation can aggravate a nematode problem. The trap cropsare special and unique varieties of white mustard and fodder radish. Theroots of the trap crops produce chemicals that stimulate nematode cysthatching in the soil. Once the nematodes hatch, they migrate to thegrowing roots of the trap crops. Once inside the roots, the nematodesstart to feed. However, the trap crops have been bred to provide thenematode with inadequate nutrition to mature, and the nematodereproductive cycle is broken.

A further reduction of the number of cyst nematodes can be achieved bystimulating parasites that attack the nematodes. Decomposed trap cropscan stimulate the development of saprophytic fungi that parasitize theeggs and nematodes within the cyst.

However, sophisticated plant breeding has now led to the development ofnew varieties that, although they attract them, are unsuitable hosts forcyst nematodes. Cultivation of these varieties can lead to a significantreduction in the population of cyst nematodes. For example, in Europe,trap crop use has increased sugar beet yields up to 33%, and increasedsugar content 8%.

By inserting trap crops into the rotation, nematode levels have beenreduced between 40 and 75%, if the crops are established at a favorabletime for the nematodes to hatch (optimum temperature is 69/70° F.).

Exemplary trap crops include, but are not limited to, solanaceous cropssuch as potato, tomato, tobacco, and other Solanum spp. such as S.sysimbriifolium, S. aethiopicum, S. quitoense, S. lycopersicoides, S.cercifolium; cruciferous crops such as radishes and mustards including,but not limited to, Brassica juncea, Sinapis alba, Raphanus sativus,Brassica napus; grain crops such as Chenopodium quinoa, Lupinusmutabilius; tuber-forming, non-Solanum spp. such as Ullucus tuberosum;and any combination thereof. In certain embodiments, the trap crop is orcomprises Solanum sysimbriifolium, particularly for potato cystnematodes.

VI. Nematicides

Nematicides are pesticides, typically synthetic pesticides, that killparasitic nematodes. Nematicides tend to be broad-spectrum toxicantsthat have properties, such as high volatility, that enable them tomigrate through the soil. Certain nematicides are carbamates, such as2-Methyl-2-(methylthio)propanal O-(N-methylcarbamoyl)oxime (Aldicarb),2,2-Dimethyl-2,3-dihydro-1-benzofuran-7-yl methylcarbamate (carbofuran),Methyl 2-(dimethylamino)-N-[(methylcarbamoyl)oxy]-2-oxoethanimidothioate(oxamyl), 2-Methyl-2-(methylsulfonyl)propionaldehydeO-(methylcarbamoyl)oxime (aldoxycarb), methyl dithiocarbamate (metamsodium).

Other nematicides are organophosphates, such as O,O-DiethylO-[4-(methylsulfinyl)phenyl] phosphorothioate (fensulfothion),1-(ethoxy-propylsulfanylphosphoryl)sulfanylpropane (ethoprop),(RS)-N-[Ethoxy-(3-methyl-4-methylsulfanylphenoxy)phosphoryl]propan-2-amine(fenamiphos),(RS)-S-sec-Butyl-O-ethyl-2-oxo-1,3-thiazolidin-3-yl-phosphonothioat(fosthiazate), S,S-di-sec-butyl O-ethyl phosphorodithioate (cadusafos).

Other nematicides include, but are not limited to, Streptomyces lydicusWYEC 108, Dimethyl N,N′[thiobis[(methylimino)carbonyloxy]]bis[ethanimidothioate]/1-[(6-Chloro-3-pyridinyl)methyl]-N-nitro-2-imidazolidinimine]combination (Thiodicarb/imidacloprid, sold as Aeris™), Bacillus firmus,chitin+urea (ClandoSan), S-Methyl 1,2,3-benzothiadiazole-7-carbothioate(Acibenzolar-S-methyl), Azadirachtin (Dimethyl(2aR,3S,4S,R,S,7aS,8S,10R,10aS,10bR)-10-(acetyloxy)-3,5-dihydroxy[(1S,2S,6S,8S,9R,11S)-2-hydroxy-11-methyl-5,7,10-trioxatetracyclo[6.3.1.0^(2.6).0^(9.11)]dodec-3-en-9-yl]-4-methyl-8-[-{(2E)-2-methylbut-2-enoyl]oxy}octahydro-1H-furo [3′,4′:4,4a]naphtho[1,8-bc]furan-5,10a(8H)-dicarboxylate), Myrothecium verrucariafermentation products (DiTera), Paecilomyces lilacinus,(RS)-S-sec-butyl-O-ethyl-2-oxo-1,3-thiazolidin-3-ylphosphonothioate(fosthiazate), Quillaja saponaria saponins (Nema-Q),5-chloro-2-(3,4,4-trifluorobut-3-ene-1-sulfonyl)-1,3-thiazole(Fluensulfone, Nimitz), ethylene dibromide, 1,2-dibromo-3-chloropropane,methyl bromide, chloropicrin, 3,5-Dimethyl-1,3,5-thiadiazinane-2-thione(dazomet), sodium tetrathiocarbonate, dimethyl dibromide, telone, orBrassica juncea extract, such as 2-propenyl isothiocyanate. A person ofordinary skill in the art will appreciate that these nematicides may beused singly, or in any combination, to effectively treat or control anematode infection. In some embodiments, certain nematicides, forexample, Brassica juncea extract, Streptomyces lydicus WYEC 108,Bacillus finnus, chitin+urea, or Paecilomyces lilacinus might besuitable for use in organic agriculture.

VII. Applications

4-Hydroxybenzyl alcohol can be used to control, such as reduce orprevent, nematode infections in commercial crops. The 4-hydroxybenzylalcohol typically is used in combination with a nematicide, a hatchingfactor, a trap crop, or a combination thereof. When used with anematicide and/or a hatching factor, the 4-hydroxybenzyl alcohol isadministered prior to, or concurrently with, administration of anematicide and/or hatching factor. In embodiments where the4-hydroxybenzyl alcohol is used in combination with both a hatchingfactor and a nematicide, the 4-hydroxybenzyl alcohol may be administeredprior to both the nematicide and hatching factor, concurrently with ahatching factor, concurrently with a nematicide, or concurrently withboth a hatching factor and a nematicide. The nematicide is administeredaccording to the manufacturers directions.

The 4-hydroxybenzyl alcohol may be applied to the soil in aconcentration suitable to facilitate and/or enhance the effect ofhatching factor(s) and/or nematicide(s). In some embodiments, theconcentration of the 4-hydroxybenzyl alcohol is from greater than zeroto 6,000 μmol/mL or more, such as from 20 μmol/mL to 2,000 μmol/mL, orfrom 100 μmol/mL to 1,000 μmol/mL. The 4-hydroxybenzyl alcohol can beapplied to soil at any suitable level selected to control nematodes,from greater than zero to 100 lbs or more per acre, such as from 1lb/acre to 100 lbs/acre, from 5 lbs/acre to 75 lbs/acre, from 20lbs/acre to 50 lbs/acre, or from 25 lbs/acre to 40 lbs/acre. In someembodiments, about 30 lbs/acre 4-hydroxybenzyl alcohol is applied to thesoil.

The 4-hydroxybenzyl alcohol may be particularly formulated for soilapplication. One disclosed formulation embodiment comprises a solutionof 4-hydroxybenzyl alcohol in a suitable solvent, such as water or anaqueous buffer solution, such as a phosphate, carbonate or acetatebuffer. Alternatively, or additionally, the formulation may compriseclays, emulsifiers, solvents, surfactants, and/or binding agents. Theformulated product may be encapsulated to form a slow release material.The formulated product may be a sprayable material in the form of awettable powder or water dispersible granule. It may be formulated as aliquid either to be applied directly to soil, applied throughchemigation, or as a sprayable product in the form of a solubleconcentrate, emulsifiable concentrate, microemulsion, oil dispersion, ormicroencapsulated particle. It may be formulated as a dry, spreadablegranule on an inert or fertilizer carrier. It may be combined withantimicrobials or other biological inhibitors. Pigments or colorants mayalso be added.

In some embodiments, the 4-hydrozybenzyl alcohol is administered to soilcontaining nematode eggs and/or cysts in the presence of a trap crop,such as within a rooting area of a trap crop. The rooting area of thetrap crop may be the area of soil around the plant into which the hatingfactor will be released by the roots. In some embodiments, the4-hydrozybenzyl alcohol is administered to soil from greater than zeroto 3 feet or more of the plant, such as from greater than zero to 2feet. The trap crop may be a trap crop that produces a hatching factor,and in some embodiments, administration of the 4-hydroxybenzyl alcoholstimulates or enhances hatch of the nematodes, which are subsequentlyattracted to the trap crop. In some embodiments, the trap crop isselected to be unsuitable for nematode reproduction, thereby reducingthe number of nematodes in the soil.

In some embodiments, the 4-hydroxybenzyl alcohol is administered incombination with a hatching factor. The 4-hydroxybenzyl alcohol may beadministered substantially simultaneously with the hatching factor, orthe hatching factor may be administered from greater than zero to 6months or more after administration of the 4-hydroxybenzyl alcohol, suchas from greater than zero to 6 months, from greater than zero to 4months, from 1 day to 2 months, from 1 day to 1 month, from 1 day to 2weeks, from 1 day to 1 week, or from 1 day to 5 days after4-hydroxybenzyl alcohol administration. In some embodiments, the4-hydroxybenzyl alcohol is administered in combination with a hatchingfactor to stimulate hatch of the nematode eggs and/or cysts when thereis no crop suitable to facilitate reproduction of the nematodes. Thisprocess effectively reduces the nematode population in soil that isadministered the combination. The 4-hydroxybenzyl alcohol/hatchingfactor may be administered to soil that has no crop planted, or it maybe administered to soil that has a trap crop growing.

In some embodiments, the 4-hydroxybenzyl alcohol is administered incombination with a nematicide. The 4-hydroxybenzyl alcohol may beadministered substantially simultaneously with the nematicide, or thenematicide may be administered from greater than zero to 6 months ormore after administration of the 4-hydroxybenzyl alcohol, such as fromgreater than zero to 6 months, from greater than zero to 4 months, from1 day to 2 months, from 1 day to 1 month, from 1 day to 2 weeks, from 1day to 1 week, or from 1 day to 5 days after 4-hydroxybenzyl alcoholadministration. Without being bound to a particular theory,administration of the 4-hydroxybenzyl alcohol may increase permeabilityof the cyst and/or egg outer membrane such that nematicide uptake isenhanced, thereby enhancing the effectiveness of the nematicide.

In some embodiments, the nematicide may be administered in combinationwith a 4-hydroxybenzyl alcohol/hatching factor combination as disclosedherein. The 4-hydroxybenzyl alcohol/hatching factor combination maystimulate hatch of the nematodes, which are then susceptible tonematicide administration. In such embodiments, the nematicide may beadministered substantially simultaneously with the hatching factor, orit may be administered subsequently to administration of the hatchingfactor, such as from greater than zero to 6 months, from greater thanzero to 4 months, from 1 day to 2 months, from 1 day to 1 month, from 1day to 2 weeks, from 1 day to 1 week, or from 1 day to 5 days afterhatching factor administration.

VIII. Examples Example 1

Hydrolysis solution composition, time, and amount of mustard meal as asource of myrosinase were varied to determine the maximum production ofallyl isothiocyanate and ionic thiocyanate from sinigrin, and sinalbin,respectively.

A. Materials and Methods 1. Materials

Mustard seeds of S. alba (IdaGold variety) and B. juncea (Pacific Gold)were obtained locally. Oil contents of seeds and meals were analyzedgravimetrically after extraction with hexane. A sinigrin standard andallyl isothiocyanate were purchased from Sigma-Aldrich (St. Louis, Mo.,USA). Standard of sinalbin was isolated from S. alba in our laboratory.Acetonitrile, water, methanol, and other solvents were of HPLC or LC/MSgrade. Solvents and all other chemicals (at least of analytical grade)were purchased from Sigma-Aldrich or ThermoFisher (Pittsburgh, Pa.,USA).

2. Mustard Meal Crude Extract Preparation

Mustard meal was homogenized and ground to a fine powder. Seed meal wasextracted with 73% (v/v) methanol at 1:20 v/v ratio using an end-to-endshaker at room temperature for 2 hours. Seed debris was separated byfiltering, and filtrates were concentrated by rotary evaporation toremove most of the solvent. Concentrated extract was then freeze-driedto obtain a free flowing powder. The concentration of sinalbin in S.alba mustard extract was 777 μmole g⁻¹ extract and the concentration ofsinigrin in B. juncea mustard extract was 555 μmole g⁻¹ extract.

3. Hydrolysis of Mustard Meal Crude Extract

Hydrolysis of mustard extracts was performed by adding correspondingmustard meal to mustard meal extract powder and then letting ithydrolyze in aqueous solution. Hydrolysis optimization was performedusing 0.1 g of mustard meal with 0.05-0.3 of extract in 2.5 mL ofaqueous solution. Hydrolysis media was modified with buffers atdifferent pH and concentrations. Time of hydrolysis was optimized from30 minutes to 48 hours under static conditions at room temperature.

4. Derivatization of Allyl Isothiocyanate

An aliquot (10-100 μL) of the hydrolysis mixture was diluted withmethanol to 5 mL. Diluted solution (860 μL) was added to 2-mL autosamplevial containing 860 μL of 100 mM potassium phosphate at pH 8.5. Then 280μL of 35 mM 1,2-benzenedithiol/1% mercaptoethanol in methanol was added,the vial was capped and incubated for 1 hour at 65° C. After incubation,mixture was vortexed, centrifuged at 24000 rpm and analyzed by HPLC/UV.

5. HPLC/UV Analysis of Derivatized Allyl Isothiocyanate

Analysis of derivatized allyl isothiocyanate was performed using anAgilent 1200 Series HPLC system with a diode array detection (DAD)system on Agilent XDB C18 (1.8 μm, 4.6×50 mm) column (Agilent, SantaClara, Calif., USA). Column was thermostated at 30° C. Isocratic elutionwas used with 90% acetonitrile in water. Flow rate was 0.6 mL/min.Spectra were recorded from 190 to 400 nm with 2 nm step. Injectionvolume was 5 μL. The runtime was 5 minutes with a derivatized allylisothiocyanate elution time of 1.4 minutes. Derivatized allylisothiocyanate was quantified at extracted wavelength channel of 350-360nm. An external calibration curve was used for quantification.

6. Ion Chromatographic Analysis

Sinigrin, sinalbin, sulfate, and ionic thiocyanate in extracts werequantified by ion chromatography (IC). IC analysis was performed using aDionex Ion Analyzer equipped with a GP40 gradient pump, ED40electrochemical detector, and an AS40 autosampler. Dionex 4×210 mmIon-Pac AS16 anion exchange column was used for separation. Sodiumhydroxide (100 mM) was used as the mobile phase at flow rate of 0.9mL/min. The detector stabilizer temperature was set at 30° C. withtemperature compensation of 1.7% per ° C. Anion suppressor current wasset to 300 mA. The injection volume was 20 μL.

7. Data Analysis

All experiments were performed at least in triplicate and are presentedas means ±one standard deviation. Significant differences among analyteconcentrations detected by different methods of analysis were determinedusing one-way analysis of variance (ANOVA) with a p<0.05 level ofsignificance. All analyses were performed using JMP software (version10, SAS Institute Inc., Cary, N.C., USA).

B. Results and Discussions 1. Optimization of Hydrolysis pH andBuffering System

During sinalbin and sinigrin enzymatic hydrolysis, several hydrolysisproducts are released (Scheme 2). Sinigrin is hydrolyzed to equimolaramounts of allyl isothiocyanate, sulfate, glucose, and hydronium ion.Hydrolysis of sinalbin leads to equimolar amounts of 4-hydroxybenzylalcohol, ionic thiocyanate, sulfate, glucose, and two moles of hydroniumion. The hydrolysis reaction is catalyzed by myrosinase enzyme, which isnaturally present in mustard. To aid hydrolysis of mustard extracts,mustard meal was added as a source of myrosinase to the hydrolysismixture. Mustard meal has relatively high mucilage content and can swellup to 400% in aqueous media. Thus the amount of meal added forhydrolysis of mustard extracts cannot exceed 20% by weight to allow forrecovery of liquid and should not exceed 4% to allow for reasonablerecovery of glucosinolates. When 0.1 g of mustard meal is added to 0.15g of mustard extracts reconstituted in 2.5 mL of water, more than 84% ofsolution can be recovered after mustard meal swelling. If higherrecoveries of glucosinolates are desired in the liquid phase, morediluted solutions of mustard extracts can be used.

For hydrolysis of endogenous glucosinolates the buffering capacity ofmustard meal is sufficient to maintain pH even when endogenousglucosinolates are hydrolyzed in the presence of water and hydronium ionis released. However, unlike endogenous concentrations in mustard meal,concentration of glucosinolates in mustard extracts are significantlyhigher. The excess of glucosinolates relative to the meal leads to thechange in pH that exceeds buffering capacity of the meal. Myrosinase hasmaximum of activity at pH of 5-7, while its activity is almostnegligible at low pH. Indeed, when the amount of mustard extract wasincreased relative to the meal, the incomplete hydrolysis was observedwith the increase of the total glucosinolate amount (FIGS. 4 and 5 ).Despite the increase of sinalbin extract added to the reaction mixture,the maximum concentration of SCN⁻ produced was leveled out at 24 mM,which is about five times higher concentration that could be producedfrom the original mustard meal. For sinigrin, a similar trend wasobserved. The maximum concentration of allyl isothiacyanate produced was14 mM even when up to 42 mM of sinigrin was added to the meal in theform of a mustard extract.

The incomplete hydrolysis of glucosinolates in mustard extracts is dueto the decrease of reaction mixture pH (FIGS. 4 and 5 ). Upon hydrolysisof endogenous sinalbin, pH typically decreases by one unit from 5.8 to4.6, at which myrosinase activity is still adequate. However, whenmustard extracts are added to the meal, more than three-fold increase ofthe sinalbin concentrations resulted in pH decrease to one more unit pH.Sinalbin concentrations four times higher than the endogenousconcentrations resulted in the pH of 2.5 and the myrosinaseinactivation. Similarly, pH of sinigrin hydrolysis mixture is decreaseto 4.6.

To prevent inactivation of myrosinase by increased acidity, a series ofphosphate buffers in the pH range from 6.0 to 7.5 was used instead ofwater for glucosinolate hydrolysis (FIG. 6 ). When 200 mM phosphatebuffer was used, complete hydrolysis of sinigrin and sinalbin wasobserved in pH range from 6.0 to 7.2, while some of glucosinolates werestill unhydrolyzed when pH was increased to 7.5. The minimumconcentration of phosphate buffer required for maintaining pH wasinvestigated and accounted for 1.5-2 times of the expected concentrationof glucosinolates in the extracts.

Other buffering agents (carbonate and bicarbonate) at the sameconcentration were equally efficient in maintaining hydrolysis mixturepH at 6.5 and providing complete hydrolysis of sinalbin and sinigrin.The use of carbonate for pH adjustment allows for the development of theglucosinolate extract pesticide which can be certified as organic andmay make the final product less expensive.

2. Optimization of Hydrolysis Media Composition

To achieve quantitative conversion of intact glucosinolates to theirbiologically active products, hydrolysis media composition was furtheroptimized. In the presence of buffer with mustard meal as a myrosinasesource, sinigrin and sinalbin are completely hydrolyzed, however only90% of corresponding biologically active hydrolysis products areproduced.

When ascorbic acid was added to the reaction mixture, almostquantitative release of allyl isothiocyanate and ionic thiocyanate wasobserved. Ascorbic acid acts as a co-factor for myrosinase and it isnaturally present in mustard meal. However, with high glucosinolateconcentrations present in mustard extracts, additional amounts ofascorbic acid are needed. Particularly, when 0.1-50 mM of ascorbic acidwas added to the hydrolysis solution, all of sinalbin was converted toSCN⁻, and all sinigrin was converted to allyl isothiocyanate (FIG. 7 ).With respect to FIG. 7 , the mass balance closure represents thepercentage of glucosinolate converted to the biologically active allylisothiocyanate and ionic thiocyanate on a molar basis.

While it may be advantageous to maintain pH and ascorbic acid content inthe hydrolysis mixture, it is also useful to carefully select mustardmeal that will be used a source of myrosinase to assure high myrosinaseactivity. Mustard meal is typically obtained by cold pressing mustardseed for oil. During the pressing process, some of the myrosinase can bedeactivated due to the local heat in the press. In fact, it has beenestimated that myrosinase activity in some processed meals may be aslittle as less than 0.5% of the activity found in the unprocessed seed.Cold pressing and defatting with hexane to remove mustard oil does notaffect the concentrations of glucosinolates, but affect the activity ofmyrosinase. Growth, harvest, and storage conditions can also affect theactivity of the myrosinase. As a result, the amount of glucosinolateshydrolyzed is lower.

3. Optimization of Hydrolysis Time

The glucosinolate-myrosinase system is designed in such a way that theincrease of water content in the plant coupled with the seed tissuerupture lead to the immediate hydrolysis reaction.

Without being bound to a particular theory, the release of hydrolysisproducts may be a defense mechanism of mustard plants. When mustardextract is hydrolyzed under static conditions, it can take a substantialperiod of time for complete hydrolysis of glucosinolates, due to thesignificant higher concentrations of glucosinolates.

Using phosphate and bicarbonate as buffering agent at finalconcentration of 150 mM, complete hydrolysis of sinalbin and sinigrinwas observed in 24 hours (FIG. 8 ). Phosphate buffer allows for fasterhydrolysis and all glucosinolates can be hydrolyzed under staticcondition in just 12 hours. Original pH of phosphate buffer is 6.5 andit coincides with the optimum pH for myrosinase. When potassiumbicarbonate was used for maintaining pH, original pH was 9.5 and thenreduces to 6.5 over the time as hydronium ions were released fromglucosinolates. Since myrosinase activity at pH 9.5 is lower than thatat pH 6.5, initial hydrolysis reaction rates were slower as compared tothe phosphate buffered systems. When no buffering agent was used,hydrolysis rates were generally slower and incomplete hydrolysis wasobserved even after reaction time of 36 hours.

Faster release of biologically active compounds can shorten bioherbicidepreparation time. However, slower release of biologically activecompounds may be beneficial for better control of pests. For example,when sinigrin is hydrolyzed, fast release of allyl isothiocyanate mayresult in undesired loss of volatile allyl isothiocyanate. At the sametime, if allyl isothiocyanate is released slowly over the time, allylisothiocyanate has better changes to interact with potential pest andultimately lead to the more efficient pest control.

Example 2

This example provides detail concerning seed meal preparation,determination of glucosinolate concentrations in defatted meal, releaseof 4-hydroxybenzyl glucosinolate from meal, and ionic thiocyanateproduction from 4-OH benzyl isothiocyanate.

All analyses and experiments were performed with meal remaining afterseed from the S. alba cultivar IdaGold was cold pressed to removeapproximately 90% of the oil. The remaining oil was removed byperforming three extractions with petroleum ether that involved shaking500 grams of the meal with 500 milliliters of petroleum ether andfiltering through a Büchner funnel. The final filtration cake was washedwith 250 milliliters of petroleum ether, allowed to air dry, andhomogenized in a blender.

Sinalbin Content of the Meal. The glucosinolate concentration of thedefatted meal was determined using a method similar to that of theInternational Organization of Standardization. Defatted seed meal wasweighed (200 mg) into 15-mL extraction tubes to which 500 mg of 3-mmglass beads, 10 milliliters of 70% methanol/water solution, and 100 μLof internal standard (4-methoxybenzyl glucosinolate, obtained frommeadowfoam (Limnanthes alba) seed meal) were added. The detectorresponse factor for 4-methoxybenzyl glucosinolate was determined bycomparison with known concentrations of 2-propenyl glucosinolate havingan assumed response factor of 1.0. Extraction tubes were shaken for 2hours on a reciprocal shaker and centrifuged for 5 min at 1073 g toprecipitate the seed meal. The extract solution was transferred tocolumns containing 250 mg of DEAE anion exchanger and allowed to drainfreely. The columns were washed twice with 1 milliliter of deionizedwater and finally with 1 milliliter of 0.1 M ammonium acetate buffer (pH4.0). To the columns was then added 100 μL of a 1 mg/L sulfatase enzyme(Sigma-Aldrich, St. Louis, Mo.) solution and 100 μL of 0.1 M ammoniumacetate buffer (pH 4.0). The columns were covered to prevent evaporationand allowed to stand with the enzyme for 12 hours, after which time thesamples were eluted into HPLC autosampler vials with two consecutive750-μL volumes of deionized water.

A Waters 2695 HPLC separation module coupled with a Waters 996photodiode array detector (PDA) and Thermabeam Mass Detector (TMD) wasused for glucosinolate analysis. For quantitative purposes alldesulfoglucosinolates detected by PDA were measured at a wavelength of229 nanometers. Separation was performed on a 250×2.00 mm, 5 μ, 125 ÅAqua C18 column (Phenomenex, Torrance, Calif.). The flow rate was 200μL/min, with a methanol gradient starting at 0.5% and increasing to 50%.Glucosinolates were identified using a combination of expected retentionbehavior (time, sequence) and mass spectra.

4-Hydroxybenzyl Isothiocyanate Release from S. alba Seed Meal. Ten gramsof the defatted meal were weighed into polypropylene centrifuge tubes towhich was added 40 mL of deionized water. In one set of triplicatesamples we added 10 milliliters of ethyl acetate as the extractant and 1μL of decane (Sigma-Aldrich, St. Louis, Mo.) as the internal standardimmediately after mixing the meal with deionized water. The mixtureswere shaken, maintained at 22±2° C., and samples removed periodicallyduring a 96-hour incubation period. In a second set of triplicatesamples, the addition of 10 milliliters of ethyl acetate and 1 μL ofdecane were delayed until 30 minutes prior to each respective samplingtime. At each sampling time the mixture was centrifuged for 10 minutesat 1677 g and 250 μL of the supernatant was withdrawn for analysis.GC-MS analysis was performed using an HP 5890A gas chromatographequipped with a 30 m×0.32 mm i.d., 0.25 pm film HP-5MS capillary column(Agilent Technologies) coupled to an HP 5972 mass detector. Ethylacetate extracts were manually injected into a split/splitless port(250° C., 20 s split) and temperature of the GC oven was programmed from65° C. (isocratic 3 minutes) to 270° C. (isocratic 5 minutes) at a rateof 15° C./minute. Average linear flow rate of helium at 250° C. was 35centimeters/minute. Data (total ion current) were corrected using decaneas the internal standard and quantified using benzyl isothiocyanate asan external standard.

Extraction efficiencies for 2-propenyl, butyl, benzyl, and t-octylisothiocyanates were determined by combining 10 μL of each in duplicate40-milliliters deionized water samples. The samples were treated in thesame manner as described above including both the immediate and delayedaddition of ethyl acetate and decane. The amount of each analyteextracted using continuous or periodic extraction was determined usingGC-MS as described for S. alba seed meal.

Stability of 4-Hydroxybenzyl Isothiocyanate in Buffered Media. Partiallypurified 4-hydroxybenzyl isothiocyanate was prepared by suspending 500grams of S. alba seed meal in 2 liters of deionized water and extractingthe mixture with 500 milliliters of ethyl acetate for 24 hours. Theethyl acetate extract was separated by decanting the top organic layerafter centrifugation, dried with 100 g of anhydrous sodium sulfateovernight, and concentrated under vacuum at laboratory temperature. Thecrude 4-hydroxybenzyl isothiocyanate extract was further purified bypreparative column chromatography on silica gel (500 grams). Elution wasachieved in a stepwise fashion using six 100-milliter aliquots of eluentcomposed of pentane and methylene chloride at ratios of 100:0, 80:20,60:40, 40:60, 20:80, and 0:100. Content of 4-hydroxybenzylisothiocyanate within the fractions was verified by GC-MS usinginstrumentation and conditions as described previously. Fractionscontaining 4-hydroxybenzyl isothiocyanate were combined and concentratedunder vacuum at laboratory temperature producing a yellowish, viscousfluid displaying only 4-hydroxybenzyl isothiocyanate andpentane/methylene chloride solvent peaks in the GC chromatogram. Nofurther concentration of 4-hydroxybenzyl isothiocyanate was achievedusing vacuum distillation because of its instability.

The pH stability of 4-hydroxybenzyl isothiocyanate was analyzed byincubating 25 μL of partially purified extract dissolved in 25milliliters of eight different buffers with pH values ranging from 3.0to 6.5 (FIG. 2 ). 0.1 M buffers were used, and were prepared by mixing0.2 M sodium citrate and citric acid solutions in pre-calculated ratiosranging from 4 milliliters sodium citrate and 46 milliliters citric acidto 41 milliliters sodium citrate and 9 milliliters citric acid in atotal volume of 100 milliliters. Actual pH values of the buffers of3.03, 3.52, 4.02, 4.49, 5.00, 5.46, 5.91, and 6.52 were verified usingan Orion model 420 A pH meter (Orion Research, Boston). At specifictimes during the incubation a 1- milliliter sample was withdrawn fromthe buffered reaction solution with a syringe and injected into a WatersIntegrity HPLC system (2695 separation module, 996 PDA, and TMD)equipped with a 150×2 mm i.d., 5 μm Aqua C-18 column (Phenomenex). Theinstrument was operated at a constant flow rate of 200 μL/min with agradient from 5 to 35% of methanol during each 30-minute run. Half-livesfor 4 hydroxybenzyl isothiocyanate were estimated from straight linesobtained by plotting the natural logarithm of the normalizedconcentration versus time (FIG. 2 ). This experiment was repeated twicewith two different meal extracts acquired by the same procedures fromthe same seed material. Half-lives from only one of the experiments arereported since the results for both experiments were similar.

Release of SCN⁻ from S. alba Seed Meal. Ten grams of defatted S. albameal were weighed into a 250-mL, polyethylene bottle to which was added200 milliliters of deionized water or a citrate buffer solution (pH of4.0, 5.0, 6.0, or 7.0) prepared as described previously. The sampleswere placed on a reciprocating shaker for 48 hours during which time5.0-milliliter aliquots were removed periodically to determine the timecourse of SCN⁻ release. Each 5-milliliter aliquot was placed in a50-milliliter centrifuge tube and 40.0 milliliters of amethanol:deionized water (2:1, v:v) solution containing 1% acetic acidwas added. The tubes were shaken vigorously for 15 minutes, centrifugedfor 5 minutes at 1073 g, and 5 milliliters of the supernatant filteredthrough a 25-mm, 0.2-μm GD/X membrane (Whatman) into a beaker. Onemilliliter of the filtered sample was then transferred to an HPLCautosampler vial to which was added 0.50 milliliter of a 0.01 M Fe³⁺solution and 100 μL of a 0.1 M HCl solution. The vials were capped,shaken, and immediately analyzed using a Waters Integrity HPLC systemequipped only with a 5-μm, 10×2 mm i.d. Aqua C-18 pre-column(Phenomenex). A 50-μL sample was injected and isocratically eluted usinga 10% methanol solution pumped at a flow rate of 0.5 milliliter/minute.Absolute concentrations of SCN⁻ in the unknown samples were determinedfollowing the same procedure as described above, except that 10.0 gramsof S. alba meal from which the glucosinolates had been removed withrepeated methanol extraction was substituted for the unaltered meal.Amounts of a KSCN stock solution containing 10 to 100 μmol of SCN⁻ wereadded to the meal/buffer mixtures prior to the initial shaking and aseparate standard curve prepared for each buffer pH (FIG. 9 ).

Glucosinolates in S. alba Meal. As expected, sinalbin was the majorglucosinolate in S. alba meal, constituting approximately 93% of totalglucosinolate content. The measured concentration of sinalbin indefatted meal was 152±5.2 μmol/gram (mean value ±variance of fivereplicates). The meal also included (2R)-2-hydroxybut-3-enylglucosinolate (3.6 μmol/g) and five unidentified glucosinolate peakswith a total estimated glucosinolate concentration of approximately 6.4μmol/g. Concentrations of indolyl glucosinolates that could potentiallyproduce SCN⁻ as a result of hydrolytic instability of their respectiveisothiocyanates represented a total of only about 1 μmol/g of defattedseed meal. Simplicity of the glucosinolate profile in S. alba meal thusfacilitates our ability to determine a likely precursor forglucosinolate hydrolysis products that might be identified. Mostimportant is the fact that low concentrations of indolyl glucosinolateseliminate the possibility that these compounds can serve as precursorsof significant amounts SCN⁻ that might be measured in hydrolyzedextracts.

4-Hydroxybenzyl Isothiocyanate Release from S. alba Seed Meal. Adramatic difference was observed between the relatively high yield of4-hydroxybenzyl isothiocyanate obtained by continuously extracting intoethyl acetate as compared to periodic measurements made by adding ethylacetate 30 minutes prior to each respective sampling time (FIG. 10 ).Maximum 4-hydroxybenzyl isothiocyanate extracted during the continuousprocedure was 162 μmol/gram seed meal at 24 hours, whereas less than 10μmol/gram was extracted at any one time in the periodic analyses. Incontrast, when continuous and periodic extractions were performed withbenzyl isothiocyanate, comparable concentrations of the compound weremeasured in the ethyl acetate extracts irrespective of the procedure.2-Propenyl, butyl, and t-octyl isothiocyanates showed extraction yieldssimilar to that of benzyl isothiocyanate ranging from at least 98% forall isothiocyanates in the continuous extraction to a low of 83% for2-propenyl isothiocyanate in the periodic extraction.

These results establish that 4-hydroxybenzyl isothiocyanate is unstablein aqueous media, and that isolation and purification require the use ofnon-reactive solvents.

Stability of 4-Hydroxybenzyl Isothiocyanate in Buffered AqueousSolutions. Partially purified and concentrated seed meal extractscontaining 4-hydroxybenzyl isothiocyanate were dissolved in buffersranging from pH 3.0 to 6.5. The half-life of 4-hydroxybenzylisothiocyanate at pH 6.5 was the shortest at 6 minutes, increasing to16, 49, 100, 195, 270, 312, and 321 minutes with decreasing pH values of6.0, 5.5, 5.0, 4.5, 4.0, 3.5, and 3.0, respectively (FIG. 2 ).Hydrolytic instability of 4-hydroxybenzyl isothiocyanate, especially athigher pH values, explains its low extractability in unbuffered extractsof seed meal that had a pH of 5.3 and a sampling time of 48 hours.Appreciable hydrolysis occurs at pH values as low as 3.0 and in a soilenvironment buffered at pH values typically between 5 and 7, significantamounts of SCN⁻ production are expected in a relatively short timeperiod.

Ionic Thiocyanate Release from S. alba Seed Meal. S. alba seed meal wasincubated with deionized water and buffer solutions ranging from pH 4.0to 7.0 to quantify SCN⁻ production resulting from 4-hydroxybenzylglucosinolate hydrolysis in the presence of a full component of mealconstituents (FIG. 9 ). SCN⁻ production occurred most slowly at pH 4.0,but final concentrations determined at 48 hours varied from a low at pH6.0 of 143 and a high in deionized water of 166 μmol/gram seed meal. Theamount of SCN⁻ expected based on 4-hydroxybenzyl glucosinolateconcentration in the meal and the assumption of its completestoichiometric conversion to SCN⁻ is approximately 152 μmol/g seed meal,thus indicating near complete conversion in 48 hours at all pH values.

Results obtained with seed meal incubations confirm conclusions reachedusing 4-OH benzyl glucosinolate extracts, clearly indicating that4-hydroxybenzyl isothiocyanate is rapidly hydrolyzed to SCN⁻ at pHvalues expected in most soils. In contrast, data from previousinvestigations conducted with purified sinalbin and myrosinase indicatethat decreased pH values promote the formation of 4-hydroxybenzylcyanide at the expense of 4-hydroxybenzyl isothiocyanate, therebydecreasing subsequent formation of SCN⁻ by approximately 50% at pH 3.0as compared to pH 7.0. The presence of additional meal componentsmoderates the influence of pH on the production of 4-hydroxybenzylcyanide, thus preserving SCN⁻ formation. Application of S. alba seedmeal to soil with the addition of sufficient water to promoteglucosinolate hydrolysis is expected to produce an amount of SCN⁻stoichiometrically equivalent to the amount of 4-hydroxybenzylglucosinolate within the meal.

SCN⁻ production in soils amended with S. alba seed meal has significantconsequences with respect to phytotoxicity and the use of meal as abioherbicide. The herbicidal activity of SCN⁻ is well known andcommercial formulations containing NH₄SCN have been marketed. Amendmentrates necessary for weed control have been determined by a number ofinvestigators for NH₄ ⁺, K⁺, and Na⁺ salts with complete removal of allvegetative cover reportedly occurring for a period of 4 months when SCN⁻was applied at rates of 270 to 680 kg/ha. Higher rates of 1,366 kgSCN⁻/ha were necessary for complete plant kill for 4 months, but a largepercentage of the weeds were removed with only 137 kilograms SCN⁻/ha.Application rates were that might alter wheat germination, and it wasfound that 342 kilograms SCN⁻/ha caused inhibition, but that the effectwas no longer observed at 69 days post application. Solutions of SCN⁻sprayed directly on vegetative growth showed that cotton defoliation waspossible using only 8.6 kilograms SCN⁻/ha.

Amounts of SCN⁻ contributed from S. alba seed meal used here, assumingcomplete stoichiometric conversion, would amount to 8.8, 17.7, and 35.3kg SCN⁻/ha for amendment rates of 1000, 2000, and 4000 kilogramsmeal/ha, respectively. Although glucosinolate concentrations in the S.alba meal used were not reported, weed control effects have beenobserved with application rates of 1000 to 2000 kilograms/ha.Phytoxicity also has been observed towards weed and crop species whenmeal was amended to greenhouse or field soils at rates from 1000 to 4000kilograms meal/ha. SCN⁻ rates provided in S. alba meal, although not ashigh as those used previously in phytotoxicity studies with solublesalts, provide SCN⁻ in amounts of potential value in weed control.

In addition to weed control benefits afforded by SCN⁻ produced as aresult of glucosinolate hydrolysis, the meals contain between 5 and 6% Nthat when mineralized represents an important nutrient source to cropplants. Organic agriculture may thus benefit from the use of S. albameal as a soil amendment both through weed control and as a nutrientsource. Potential environmental effects appear minimal given thatbiological degradation of SCN⁻ has been observed in soils and S. alba istypically grown as a condiment mustard for human consumption.

Glucosinolate concentrations in Brassicaceae seed meals as may bedetermined according to the method of this example are shown in Table 1below.

TABLE 1 Glucosinolate concentrations in Brassicaceae seed meals. B.juncea B. napus B. napus S. alba “Pacific Glucosinolate R-group “Athena”“Sunrise” “Ida Gold” Gold” μmol g⁻¹ of sample (2R)-2-hydroxy-3 - 1.5 1.33.4 0.5 butenyl 2-propenyl 0.4 123.8 (2S)-2-hydroxy-3- butenyl)2-hydroxy-4-butenyl) 0.2 1.8 (2R)-2-hydroxy-4- 0.5 pentenyl4-hydroxy-benzyl 148.1 Unknown 9.1 3-butenyl 2.8 2.7 4-hydroxy-3- 11.310.9 0.74 indolylmethyl (0.28) unknown 2.6 unknown 0.74 4-pentenyl 1.31.4 3-indolylmethyl 0.9 0.8 4-methylthiobutyl 1.7 N-methoxy-3- 0.1 0.010.6 indolylmethyl unknown 1.33 TOTAL 20.1 17.2 165.75 126.14

Highest glucosinolate concentrations were measured in S. alba IdaGoldmeal with 4-OH benzyl showing as the dominant glucosinolate. The B.juncea variety Pacific Gold had the next highest glucosinolateconcentration, with propenyl glucosinolate dominating the total. It hasbeen shown that both 4-OH benzyl and propenyl glucosinolates produce ITCas an end product of hydrolysis at typical soil pH values.

More recent evidence indicates that this assumption is not true for 4-OHbenzyl glucosinolate. ITC production is significant since this compoundis considered to be the most toxic of all glucosinolate hydrolysisproducts and thus most important in pest control. Recent results withweed seed bioassays prompted a reevaluation of this assumption andfurther prompted considering the inhibitory properties of othercompounds, such as ionic thiocyanate.

The remaining B. napus varieties, Athena and Sunrise, were included asthey routinely are used as an amendment in bioassay control experiments,and only low glucosinolate concentrations were present.

Example 3

An initial experiment was conducted to determine possible effects of S.alba seed meal on hatch of Globodera pallida eggs. Measuring thepercentage egg hatch is a viability indicator and provides an indicationof the potential impact from a treatment. In this experiment, Globoderapallida encysted eggs were exposed to S. alba seed meal for 2 weeks insmall containers containing a sand:soil mix. The sand:soil mix withoutseed meal was used as the non-treated control. After a 2-week exposure,cysts were removed from the containers, and a hatching assay wasconducted by placing eggs from each treatment into individual wells thatcontained either potato root diffusate (PRD) to stimulate hatch, or asoil extract (SE) that did not contain the hatching stimulus. Theexperiment had two treatments: 1) a non-treated bare soil control; or 2)S. alba seed meal. Each treatment included 5 replicates. Eggs from eachof these treatments were exposed to either PRD or SE for 2 weeks, atwhich time hatched infectious 2^(nd) stage Globodera pallida juveniles(J2) were counted.

As FIG. 11 indicates, Globodera pallida hatch in PRD was greater afterexposure to S. alba seed meal than hatch from the non-treated control(bare soil). As expected, hatch of the non-treated eggs was higher inPRD than from soil extract because PRD contains the hatching stimulusand soil extract does not. Percent hatch of eggs exposed to S. alba insoil extract did not differ from the non-treated eggs. But when exposedto PRD, approximately 30% more eggs hatched with prior exposure to S.alba seed meal compared to non-exposed eggs. This experiment was thefirst indication that Globodera pallida egg hatch could be enhanced byexposure to compounds found in yellow mustard seed meal.

Example 4

The objective of this experiment was to determine if 4-hydroxybenzylalcohol (HBA), the product of enzymatic hydrolysis of the glucosinolatesinalbin contained in S. alba seed meal, could be responsible for theobserved enhanced hatch. In an experiment similar to Example 1, encystedeggs were exposed to either HBA, or S. alba seed (yellow mustard) meal.The non-treated eggs were placed in bare soil mix only. After 2 weeks,exposed eggs were placed in hatching assays in either PRD or in soilextract, and hatched J2 were counted.

The results were similar to the results from Example 1 (FIG. 12 ). Inthe absence of the hatching stimulus found in PRD, percent hatch in soilextract was minimal for both non-treated eggs and eggs exposed to S.alba meal. Again, hatch of eggs exposed to S. alba meal wasapproximately 30% greater compared to non-treated control when hatchedin the presence of the hatching stimulus found in PRD. 4-hydroxybenzylalcohol had an even greater impact on hatch than S. alba meal alone; egghatch was 50% higher when exposed to HBA prior to PRD exposure, than thenon-treated control when hatched in PRD. As FIG. 12 indicates, theenzymatic hydrolysis product, 4-hydroxybenzyl alcohol, is the compoundfrom S. alba meal that enhances G. pallida hatch.

Example 5

In this experiment, the impact of different application rates of HBA onpercent Globodera pallida hatch was investigated (FIG. 13 ). Again,hatch of the non-treated control was higher in PRD than in soil extract.Exposure to HBA rates of 1 μmol/ml enhanced hatch of Globodera pallidamore effectively than higher rates tested (2 μmol/ml, 4 μmol/ml, 8μmol/ml). An increase in hatch of approximately 60% occurred with a 1μmol/ml pre-treatment with HBA prior to PRD exposure. The enhanced hatchcaused by 4-hydroxybenzyl alcohol was consistent in all experiments anddemonstrated to be effective at different rates of application.

IX. Statements

The following numbered statements illustrate exemplary embodiments ofthe disclosed technology.

Statement 1. A method for controlling nematodes, comprising:

applying 4-hydroxybenzyl alcohol, Sinapsis alba plant extract, Sinapsisalba seed meal, or a combination thereof, to soil having, or at risk ofhaving, nematode eggs, in an amount and manner sufficient to effectnematode egg hatch.

Statement 2. The method of statement 1, wherein the 4-hydroxybenzylalcohol, Sinapis alba plant extract, Sinapis alba seed meal, orcombination thereof, is applied in the presence of a trap crop.

Statement 3. The method of statement 2, wherein the trap crop is asolanaceous crop, a cruciferous crop, a grain crop, a tuber-forming,non-Solanum species, or a combination thereof.

Statement 4. The method of any one of statements 1-3, wherein the trapcrop is a potato crop, tomato crop, tobacco crop, mustard crop, radishcrop, or a combination thereof.

Statement 5. The method of any one of statements 1-3, wherein the trapcrop is S. sysimbriifolium, S. aethiopicum, S. quitoense, S.lycopersicoides, S. cercifolium, Brassica juncea, Sinapis alba, Radaphussativus, Brassica napus, Chenopodium quinoa, Lupinus mutabilius, Ullucustuberosum, or a combination thereof.

Statement 6. The method of any one of statements 1-5, wherein applyingthe 4-hydroxybenzyl alcohol, Sinapis alba plant extract, Sinapis albaseed meal, or a combination thereof, to the soil in the presence of thetrap crop comprises applying the 4-hydroxybenzyl alcohol, Sinapis albaplant extract, Sinapis alba seed meal, or combination thereof, to thesoil from greater than zero to 3 feet from the trap crop.

Statement 7. The method of any one of statements 1-6, further comprisingapplying a hatching factor to the soil.

Statement 8. The method of statement 7, wherein the hatching factorcomprises a potato hatching factor.

Statement 9. The method of statement 7 or statement 8, wherein thehatching factor comprises a potato root diffusate.

Statement 10. The method of any one of statements 7-9, wherein thehatching factor comprises a tomato root diffusate.

Statement 11. The method of any one of statements 7-10, wherein thehatching factor comprises a soybean root diffusate.

Statement 12. The method of any one of statements 7-11, wherein thehatching factor comprises a sugar beet root diffusate.

Statement 13. The method of any one of statements 7-12, wherein thehatching factor is applied substantially simultaneously with the4-hydroxybenzyl alcohol, Sinapsis alba plant extract, Sinapis alba seedmeal, or a combination thereof.

Statement 14. The method of any one of statements 7-12, wherein thehatching factor is applied from greater than zero to 6 months afterapplication of the 4-hydroxybenzyl alcohol, Sinapis alba plant extract,Sinapis alba seed meal, or a combination thereof.

Statement 15. The method of any one of statements 1-14, furthercomprising applying a nematicide to the soil.

Statement 16. The method of statement 15, wherein the nematicide is acarbamate or an organophosphate.

Statement 17. The method of statement 15 or statement 16, wherein thenematicide is 2-Methyl-2-(methylthio)propanalO-(N-methylcarbamoyl)oxime, 2,2-Dimethyl-2,3-dihydro-1-benzofuran-7-ylmethylcarbamate, Methyl2-(dimethylamino)-N-[(methylcarbamoyl)oxy]-2-oxoethanimidothioate,2-Methyl-2-(methylsulfonyl)propionaldehyde O-(methylcarbamoyl)oxime,O,O-Diethyl O-[4-(methylsulfinyl)phenyl] phosphorothioate,1-(ethoxy-propylsulfanylphosphoryl)sulfanylpropane,(RS)-N-[Ethoxy-(3-methyl-4-methylsulfanylphenoxy)phosphoryl]propan-2-amine,Streptomyces lydicus WYEC 108, Dimethyl N, N′[thiobis[(methylimino)carbonyloxy]]bis[ethanimidothioate]/1-[(6-Chloro-3-pyridinyl)methyl]-N-nitro-2-imidazolidinimine]combination, Bacillus firmus, chitin+urea, S-Methyl1,2,3-benzothiadiazole carbothioate, Dimethyl(2aR,3S,4S,R,S,7aS,8S,10R,10aS,10bR)-10-(acetyloxy)-3,5-dihydroxy-4-[(1S,2S,6S,8S,9R,11S)-2-hydroxy-11-methyl-5,7,10-trioxatetracyclo[6.3.1.0^(2.6).0^(9.11])dodec-3-en-9-yl]-4-methyl-8-[-{(2E)-2-methylbut-2-enoyl]oxy}octahydro-1H-furo[3′,4′:4,4a]naphtho[1,8-bc]furan-5,10a(8H)-dicarboxylate,

Myrothecium verrucaria fermentation products, Paecilomyces lilacinus,(RS)-S-sec-butyl-O-ethyl-2-oxo-1,3-thiazolidin-3-ylphosphonothioate,Quillaja saponaria saponins,5-chloro-2-(3,4,4-trifluorobut-3-ene-1-sulfonyl)-1,3-thiazole, Brassicajuncea extract, or a combination thereof.

Statement 18. The method of any one of statements 15-17, wherein thenematicide is applied substantially simultaneously with the4-hydroxybenzyl alcohol, Sinapis alba plant extract, Sinapis alba seedmeal, or a combination thereof.

Statement 19. The method of any one of statements 15-17, wherein thenematicide is applied from greater than zero to 6 months afterapplication of the 4-hydroxybenzyl alcohol, Sinapis alba plant extract,Sinapis alba seed meal, or a combination thereof.

Statement 20. The method of any one of statements 1-19, wherein thenematode eggs are encysted eggs.

Statement 21. The method of any one of statements 1-20, wherein thenematode egg is a potato nematode egg.

Statement 22. The method of statement 21, wherein the potato nematodeegg is the egg of a potato nematode selected from Globodera achilleae,Globodera artemisiae, Globodera chaubattia, Globodera ellingtonae,Globodera hypolysi, Globodera leptonepia, Globodera millefolii,Globodera mirabilis, Globodera pallida, Globodera pseudorostochiensis,Globodera rostochiensis, Globodera tabacum, Globodera zelandica, or acombination thereof.

Statement 23. The method of any one of statements 1-20, wherein thenematode egg is a soy nematode egg.

Statement 24. The method of statement 23, wherein the soy nematode eggis the egg of Heterodera glycines.

Statement 25. The method of any one of statements 1-20, wherein thenematode egg is a sugar beet nematode egg.

Statement 26. The method of statement 25, wherein the sugar beetnematode egg is the egg of Heterodera schachtii.

Statement 27. The method of any one of statements 1-20, wherein thenematode egg is the egg of a nematode selected from a Globodera species,Heterodera species, Meloidogyne species, Pratylenchus species, Xiphenamaspecies, or a combination thereof.

Statement 28. The method of any one of statements 1-20, wherein thenematode egg is the egg of a nematode selected from Globodera achilleae,Globodera artemisiae, Globodera chaubattia, Globodera ellingtonae,Globodera hypolysi, Globodera leptonepia, Globodera millefolii,Globodera mirabilis, Globodera pallida, Globodera pseudorostochiensis,Globodera rostochiensis, Globodera tabacum, Globodera zelandica,Heterodera carotae, Heterodera cruciferae, Heterodera humuli, Heteroderatrifolii, Heterodera galeopsidis, Heterodera goettingiana, Heteroderabetae, Heterodera sacchari, Heterodera cajani, Heterodera avenae,Heterodera filipjevi, Heterodera arenaria, Heterodera aucklandica,Heterodera bifenestra, Heterodera hordecalis, Heterodera in, Heteroderalatipons, Heterodera mani, Heterodera pratensis, Heterodera spinicauda,Heterodera turcomanica, Heterodera glycines, Heterodera schachtii, M.incognita, M. hapla, M. javanica, M. chitwoodi, M. arenaria, M. naasi,M. acronea, M. artiellia, M. brevicauda, M. coffeicola, M. exigua, M.gajuscus, M. enterolobii, M. partityla, M. thamesi, M. fallax,Pratylenchus alleni, Pratylenchus brachyurus, Pratylenchus coffeae,Pratylenchus crenatus, Pratylenchus dulscus, Pratylenchus fallax,Pratylenchus flakkensis, Pratylenchus goodeyi, Pratylenchus hexincisus,Pratylenchus loosi, Pratylenchus minutus, Pratylenchus mulchandi,Pratylenchus musicola, Pratylenchus neglectus, Pratylenchus penetrans,Pratylenchus pratensis, Pratylenchus reniformia, Pratylenchus scribneri,Pratylenchus thornei, Pratylenchus vulnus, Pratylenchus zeae, X.americanum, X. diversicaudatum, X. index, X. italiae, X. bakeri, X.brevicolle, X. insigne, X. rivesi, X. vuittenezi, X. brasiliense, X.hygrophylum, X. stenocephalum, X. elongatum, X. coxi, X. ingens, X.conurum, X. pachydennum, X. mammallatum, X. thorneanum, X. melitense, X.bacaniboia, X. surinamense, X. guirani, X. porosum, X. rotundatum, X.spinuterus, X. bergeri, X. douceti, X. nigeriense, X. attarodorum, X.clavicaudatum, X. elongatum, X. ifacolum, X. longidoroides, X. setariae,X. ifacolum, X. ebriense, X. pini, X. turcicum, X. basiria, X.marsupilami, X. malagasi, X. radicicola, X. longicaudatum, X. krugi, X.costaricense, X. filicaudatum, X. pachtaicum, or a combination thereof.

Statement 29. The method of any one of statements 1-28, wherein applyingthe 4-hydroxybenzyl alcohol, Sinapis alba plant extract, Sinapis albaseed meal, or a combination thereof to the soil comprises applying anamount of Sinapis alba seed meal of from 500 lbs/acre to 4,000 lbs/acre.

Statement 30. The method of any one of statements 1-28, wherein applyingthe 4-hydroxybenzyl alcohol, Sinapis alba plant extract, Sinapis albaseed meal, or a combination thereof to the soil comprises applying anamount of Sinapis alba plant extract of from 50 lbs/acre to 1,000/acre.

Statement 31. The method of any one of statements 1-30, comprisingapplying 4-hydroxybenzyl alcohol to the soil.

Statement 32. The method of statement 31, wherein applying4-hydroxybenzyl alcohol to the soil comprises applying an amount of4-hydroxybenzyl alcohol of from greater than zero to 100 lbs/acre.

Statement 33. The method of statement 32, wherein the amount of4-hydroxybenzyl alcohol applied to the soil is from 20 lbs/acre to 50lbs/acre.

Statement 34. The method of any one of statements 31-33, comprisingapplying 4-hydroxybenzyl alcohol to the soil in the presence of the trapcrop.

Statement 35. The method of any one of statements 31-34, comprising:

applying 4-hydroxybenzyl alcohol to the soil; and

applying a hatching factor to the soil.

Statement 36. The method of any one of statements 31-34, comprising:

applying 4-hydroxybenzyl alcohol to the soil; and

applying a nematicide to the soil.

Statement 37. The method of any one of statements 31-34, comprising:

applying 4-hydroxybenzyl alcohol to the soil; and

applying a nematicide and a hatching factor to the soil, substantiallysimultaneously, or sequentially in any order.

Statement 38. The method of any one of statements 1-37, wherein applyingthe 4-hydroxybenzyl alcohol comprises applying a formulation comprisinga concentration of 4-hydroxybenzyl alcohol of from greater than zero to6,000 μmol/mL.

Statement 39. The method of statement 38, wherein the concentration of4-hydroxybenzyl alcohol is from 100 μmol/mL to 1,000 μmol/mL.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A method for controlling nematodes, the method comprisingcontacting soil with 4-hydroxybenzylalcohol, Sinapis alba plant extract,or Sinapis alba seed meal, or a combination thereof, wherein the soilcontains or is at risk of containing nematode eggs, to thereby affectnematode egg hatch.
 2. The method according to claim 1, wherein a cropplant is being cultivated in the soil that is contacted with the4-hydroxybenzylalcohol, Sinapis alba plant extract, or Sinapis alba seedmeal, or the combination thereof.
 3. The method according to claim 2,wherein the 4-hydroxybenzylalcohol, Sinapis alba plant extract, orSinapis alba seed meal, or the combination thereof is applied to thesoil in the rooting area of the cultivated crop plant.
 4. The methodaccording to claim 2, wherein hatched nematodes migrate to the roots ofthe crop plant and die without reproducing.
 5. The method of claim 2,wherein the crop plant is a solanaceous crop plant, a cruciferous cropplant, a grain crop plant, a tuber-forming crop plants, or a non-Solanumspecies crop plant, or a combination thereof.
 6. The method of claim 2,wherein the crop plant is removed from the soil, and wherein thereafteranother crop plant is grown in the soil, wherein nematode levels in thesoil have been substantially reduced.
 7. The method of claim 6, whereinnematode levels have been substantially reduced by from about 40% toabout 75%.
 8. The method of claim 1, the method further comprisingapplying a hatching factor, a nematicide, or both to the soil.
 9. Themethod of claim 8, wherein the hatching factor comprises a potatohatching factor, a potato root diffusate, a tomato root diffusate, asoybean root diffusate, a sugar beet root diffusate, or a combinationthereof.
 10. The method of claim 8, wherein the hatching factor isapplied substantially simultaneously with the 4-hydroxybenzyl alcohol,Sinapis alba plant extract, Sinapis alba seed meal, or the combinationthereof, or the hatching factor is applied from greater than zero to 6months after application of the 4-hydroxybenzyl alcohol, Sinapis albaplant extract, Sinapis alba seed meal, or the combination thereof 11.The method of claim 8, wherein the hatching factor is applied to thesoil a distance from greater than zero to 3 feet from the crop plant.12. The method of claim 8, wherein the nematicide is appliedsubstantially simultaneously with the 4-hydroxybenzyl alcohol, Sinapisalba plant extract, Sinapis alba seed meal, or the combination thereof.13. The method of claim 8, wherein a hatching factor is appliedsubstantially simultaneously with the 4-hydroxybenzyl alcohol, Sinapisalba plant extract, Sinapis alba seed meal, or the combination thereof,and the nematicide.
 14. The method of claim 8, wherein the nematicide isapplied from greater than zero to 6 months after application of the4-hydroxybenzyl alcohol, Sinapis alba plant extract, Sinapis alba seedmeal, or the combination thereof.
 15. The method of claim 8, wherein thehatching factor is applied from greater than zero to 6 months afterapplication of the 4-hydroxybenzyl alcohol, Sinapis alba plant extract,Sinapis alba seed meal, or the combination thereof.
 16. The method ofclaim 1, wherein the nematode eggs are encysted eggs.
 17. The method ofclaim 16, wherein the nematode eggs are potato nematode eggs, soynematode eggs, sugar beet nematode eggs, or a combination thereof. 18.The method of claim 1, wherein the contacting comprises applying anamount of Sinapis alba seed meal of from about 500 lbs/acre to about4,000 lbs/acre to the soil.
 19. The method of claim 1, wherein thecontacting comprises applying an amount of 4-hydroxybenzyl alcohol offrom greater than zero to about 100 lbs/acre.
 20. The method of claim 1,wherein applying the 4-hydroxybenzyl alcohol comprises applying aformulation comprising a concentration of 4-hydroxybenzyl alcohol offrom greater than zero to 6,000 μmol/mL.